Endothelial barrier dysfunction is a hallmark of lung inflammation and the proximate cause of pulmonary edema. Endothelial barrier integrity is determined by axial contractile forces which are determined by the MLCK-actomyosin apparatus. These forces are opposed by and tethering forces comprised by cell-cell and cell-matrix adhesions that resist contraction and promote barrier integrity. Endothelial myosin light chain kinase (MLCK)-dependent contractile mechanisms are vital to edema formation, however, additional mechanisms which do not required activation of this important kinase, may be equally important in producing endothelial barrier dysfunction. For example, phorbol ester, ionomycin, and pertussis toxin induce MLCK-independent endothelial permeability. Based on our preliminary data, they will test the hypothesis that MLCK-independent endothelial cell barrier dysfunction may result in altered interactions between the endothelial cytoskeleton and tethering mechanisms involving focal adhesions and adherens junctions. SA #1 Will examine the regulation of focal adhesion plaque proteins in MLCK-independent barrier dysfunction. In addition to radiolabelling and immunoprecipitation techniques, they will utilize immunofluorescent localization and biochemical subcellular characterization of focal adhesion plaque proteins to define the relationship between focal adhesion organization, protein phosphorylation, and association with the cytoskeleton. SA #2 will examine the regulation of adherens junction proteins in MLCK-independent barrier dysfunction. Utilizing a similar approach to SA #1, the phosphorylation, histochemistry, and subcellular distribution of adherens junction proteins will be defined. SA #3 will examine the role of PKC and PKA in modulating tethering protein phosphorylation and function. Finally, utilizing standard molecular biological techniques, SA #4 will examine the molecular regulation of p125fak and VE-cadherin expression in MLCK-independent permeability. These studies will define the functional consequences of tethering force alterations by measurements of endothelial cell gap formation, force development, resistance, and permeability. The in depth analysis of novel MLCK-independent mechanisms that potentially contribute to endothelial cell barrier dysfunction, will lead to increased understanding of edema formation relevant to diverse inflammatory lung syndromes. (End of abstract)